Vaccines are a crucial public health intervention, yet to date their development has been largely ad hoc and empiric. Infections are most common at the extremes of age and vaccine adjuvantation can be a key approach to enhance immunogenicity for special populations that have distinct immunity. Adjuvants, molecules that boost immune response, can greatly enhance immunity but much remains to be learned regarding adjuvant action that can vary markedly with age. To identify promising adjuvantation systems and characterize their mechanisms of action, the Levy Lab has established novel human in vitro model systems, including whole- blood assays, monocyte-derived dendritic cell (MoDC) arrays, and microphysiologic three-dimensional tissue constructs, which model innate and adaptive immune responses. In preliminary in vitro studies employing human newborn, infant and adult leukocytes, we have identified certain Toll-like receptor agonist (TLRA) and C-type lectin receptor agonist (CLRA) combinations that synergistically enhance NF-kB and inflammasome activation as well as Th1 cytokine production in early life: the synthetic imidazoquinoline TLR7/8 agonist R848 and the Mincle (CLR) agonist Trehalose-6,6-dibehenate (TDB). In the propose studies, we will work in collaboration Dr. Peter Andersen (Statens Serum Institut (SSI); Copenhagen, Denmark), whose laboratory has world class expertise in adjuvant formulation and vaccinology, to further characterize the mechanisms underlying the synergistic activity of this combination adjuvantation system using Respiratory Syncitial Virus (RSV) pre-fusion protein as a model antigen. Our hypothesis is that TLRAs and CLRAs synergistically activate the NF-?B and inflammasome pathways to induce robust Th1 immune responses in vitro and in vivo. Our goal is to test this hypothesis employing our novel human in vitro platforms and in vivo safety immunogenicity of our adjuvant combination in using RSV pre-fusion protein as model antigen. We will achieve this goal by pursuing the following Specific Aims (SAs): Aim 1: Characterize mechanisms of combination adjuvant synergy in activating human DCs in vitro. Aim 2: Assess the ability of combination adjuvants to enhance DC-mediated lymphocyte activation in vitro. Aim 3: Evaluate the impact of combinatorial adjuvants on biomarkers of vaccine reactogenicity and immunogenicity in vitro and in vivo. Overall, successful pursuit of these Specific Aims will provide fresh insight into mechanisms of age-specific adjuvant synergy in vitro and in vivo thereby advancing new approaches to enhance protective immune responses in special populations, including the very young, against key pathogens.